Table 6 GC/MS settings

  ▲CRITICAL Prior to GC-MS analysis, ensure that the instrument has been optimized. Details for our GC/MS workflow are listed below. Please be aware that other GC/MS methods with sufficient sensitivity, reproducibility, and linearity for the metabolites of interest can be used.

 1. Connect an Agilent 5977B Mass Spectrometer to an Agilent 7890B gas chromatograph. Use 5977MS/Enhanced MassHunter software to control the GC/MS system.

  GC settings:

 Column: Agilent 19091S-433HP-5MS 5% Phenyl Methyl Silox 325 °C: 29.8 m × 250 μm × 0.25 μm

 Carrier gas: helium (>  99.999% purity)

 Carrier gas flow rate: 1 mL/min

 Oven temperature program: 100 °C for 3 min, increase the temperature by 10 °C/min to 140 °C, increase the temperature by 8 °C/min to 260 °C, increase the temperature by 10 °C/min to 310 °C, and hold at this temperature for 5 min.

 Injection port temperature: 280 °C

 Transfer line temperature: 250 °C

 Injection volume: 1 μL

 Mode: splitless

  MS settings:

 Ionization mode: electron ionization (EI) mode

 Electron energy: 70 eV

 Ion source temperature: 250 °C

 Scan frequency: 2.7/s

 Mass range: 50.00–600.00 m/z

 2. Place the samples in the auto sampler tray for GC/MS analysis.

 3. Create a sample list (or sequence list) that includes the metabolomic sample details.

 4. Inject the pyridine standard at the beginning and the end of the sample analysis sequence.